PROJECT 2: Molecular Inhibition of Apoptosis Inhibitors B-cell chronic lymphocytic leukemia [B-CLL] arises primarily because of failures in apoptosis mechanisms. Aberrant over-expression of anti-apoptotic Bcl-2 family proteins contributes greatly to the long lifespan of CLL cells, and also thwarts attempts to eradicate these leukemic cells by chemotherapy. The human genome contains six genes encoding anti-apoptotic Bcl-2 family proteins (Bcl-2, BCI-XL, Mcl-1, Bfl-1, Bcl-W, Bcl-B), several of which are often highly expressed in CLLs. Expression of Mcl-1 or Bfl-1 accounts for resistance to chemical antagonists of Bcl-2, such as ABT263 analogs. Moreover, expression of these pro survival proteins increases in CLL cells thriving in microenvironmental niches. We hypothesize that redundancy caused by multiple anti-apoptotic Bcl-2 family members is a critical barrier to effective treatment of CLL. We propose to test this hypothesis through 3 complementary approaches. First. ABT263 and other small molecule Bcl-2 antagonists currently in clinical development bind a regulatory site on Bcl-2, mimicking endogenous antagonists that contain the BH3 domain. We have generated novel BH3 mimicking compounds with broad-spectrum inhibitory activity against all anti-apoptotic Bcl-2 family proteins. These compounds will be tested for preclinical activity against primary human CLL cells in culture and against murine CLL cells in transgenic mouse models. Second, we have identified a non-BH3 regulator of Bcl-2 in the Nur77/TR3 protein, an orphan nuclear receptor that binds to Bcl-2, Bfl-1, and Bcl-B, converting these proteins from antito pro-apoptotic. Using Nur77/TR3, we have discovered a novel non-BH3 regulatory site on Bcl-2 family proteins that will be targeted with small molecules as an alternative approach to Bcl-2 antagonism. Third. expression of many anti-apoptotic Bcl-2 family proteins (including Mel-1, BCI-XL, and Bfl-1) is upregulated when CLL cells are influenced by microenvironment. Hence, agents from Aims 1 and 2 will be evaluated for activity against CLL cells using in vitro culture models of microenvironment interactions. Altogether, our goal is to extend preclinical studies of novel Bcl-2 family antagonists towards the ultimate goal of bringing these concepts and new agents into the clinic via the CLL Research Consortium (CRC).